Kinetics of decay in the expression of interferon - dependent mRNAs responsible for resistance to virus ( interferon action / antiviral kinetics / 5 , 6 - dichloro - l - # - D - ribofuranosylbenzimidazole

We used 5,6-dichloro-1-D-ribofuranosylbenzimidazole (DBB), a selective and reversible inhibitor of mRNA production; io investigate the regulation of the pathway leading-to resistance to viruses in cells treated with interferon (IF). DRB allows initiation of transcription but promotes premature termination of the nucleotide chains, so that it abolishes interferon-dependent protection against viruses. When the DRB is removed, synthesis of complete mRNAs can resume. Mouse L-929 cells were exposed to 100 AM DRE before and during a 1-hr pulse of IF followed by treatment with antibody to IF to prevent cell-to-cell spread of IF after that time. At different intervals thereafter the cells were washed and the DRB was replaced by medium; after further incubation, the cells were infected with vesicular stomatitis virus. Resistance to virus was inversely proportional to the duration of the block imposed by DRB. When the DRB was removed soon after the IF pulse, substantial protection from virus ensued, but none developed when removal of the DRB was deferred for 5-6 hr. Cells exposed to DRB for 5 hr, then pulsed with IF for 1 hr, still mounted a strong antiviral response. The data show that the ability of cells to resist viral infection decays within 5-6 hr after treatment with IF. Whether the decay is due to shutoff of transcription of mRNAs, or to their destruction or degradation, or whether regulation takes place at one or more subsequent steps in the antiviral pathway, remains to be determined. The fact that the protection against viruses conferred upon cells by exposure to homologous interferon (IF) depends upon new mRNA synthesis was inferred from experiments in which actinomycin D was found to abolish the antiviral response when it was administered at the same time as the interferon (1, 2). As the time between treatment with IF and the addition of actinomycin was extended, so that longer periods of mRNA synthesis were permitted, the antiviral response reappeared and increased, until after several hours of delay in administering actinomycin, the drug no longer blocked establishment of the antiviral state (3, 4). It was deduced that the amount of protection from viral infection is proportional to the amount of IF-dependent mRNAs made and that synthesis of a protective quantity of such mRNAs is achieved by the time that actinomycin treatment no longer prevents resistance to viruses. Experiments with enucleated cells supported these findings (5, 6), as have recent studies of enzymes that inhibit mRNA translation (7-9). They include oligoisoadenylate synthetase and eukaryotic initiation factor 2 (eIF-2) protein kinase. These enzymes, and also a 2',5'-phosphodiesterase, have recently been isolated from IF-treated cells (10-13), as has an associated mRNA (12). The enzymes display IF dose dependence similar to that shown for development of resistance to viruses (13), and the induction of the isolated IF-dependent proteins is blocked by exposure of the cells to actinomycin early after IF treatment (12-14). The experiments reported here had a different aim from The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 452 those just mentioned. The goal of our experiments was to estimate how long the cell remains committed to the expression of the IF-dependent transcriptional responses that result in virus resistance, once transcription is completely switched on through the action of transmembrane or other IF-induced signal molecules. Such experiments are now practicable with the aid of 5,6-dichloro-1-f-D-ribofuranosylbenzimidazole (DRB), a selective and reversible inhibitor of heterogeneous nuclear RNA and mRNA syntheses (15-17). Inhibition of mRNA synthesis imposed by DRB is rapid and the inhibition is promptly reversed after removal of the DRB by medium change (18). Recent studies indicate that DRB permits uninterrupted initiation of transcription but promotes premature termination of nucleotide chains (19, 20). It has only a minor effect on protein synthesis when cells are treated for several hours (15, 16, 18). We postulated that cells treated with IF would be capable of initiating transcription of IF-dependent mRNAs during a period of DRB exposure but that the premature termination of the nucleotide chains would render them incapable of coding for the respective antiviral proteins. We presumed that if the interval between treatment with IF and removal of the DRB were not excessive, complete mRNA chains would begin to appear soon after the DRB block was lifted and that this would be manifested as the acquisition of resistance to viral infection. Within limits, a subsequent decay or shutoff in the synthesis or expression of IF-dependent mRNA could also be observed if the interval between IF treatment and DRB removal were sufficiently prolonged. Data from experiments designed to test these assumptions indicate that cellular commitment to the expression of IF-dependent mRRNAs for protection against viruses does decay within a relatively few hours after the administration of a 1-hr pulse of interferon terminated by antibody to interferon, perhaps due to negative regulation of one or more steps in the antiviral pathway. MATERIALS AND METHODS Cell Cultures and Viruses. Mouse L-929 cells obtained from the American Type Culture Collection were grown in Eagle's basal medium (BME) plus 10% fetal calf serum (FCS). Coverslip monolayers used in experiments contained approximately 8 X 104 cells in BME/2% FCS. They were incubated at 370C in 35-mm plates. Like cultures in Microtiter wells, coverslip monolayers require only small reagent and inoculum volumes (0.1 ml); but unlike Microtiter cultures, the cells remain attached after repeated fluid additions and washings. Mouse C-243 cells were obtained from R. H. Bassin, National Institutes of Health. Abbreviations: DRB, 5,6-dichloro-1-f-D-ribofuranosylbenzimidazole; IF, interferon; MIF, mouse interferon; anti-MIF, antibody to mouse interferon; eIF-2, eukaryotic initiation factor 2; BME, Eagle's basal medium; FCS, fetal calf serum; VSV, vesicular stomatitis virus; NDV, Newcastle disease virus; PFU, plaque-forming units. Proc. Natl. Acad. Sci. USA 77 (1980) 453 They were cultivated in BME plus 10% heat-inactivated FCS. Vesicular stomatitis virus (VSV), Indiana serotype, was plaque purified three times in L-929 cells before a stock virus pool of a large-plaque variant was grown in secondary chicken embryo fibroblasts. The stock virus titer was 4 X 108 plaque-forming units (PFU)/ml. Newcastle disease virus (NDV) was propagated in the allantoic cavity of 10to 11-day-old embryonated chicken eggs and yielded 2000 hemagglutinating units per ml of allantoic fluid. NDV used for IF induction was pelleted by centrifugation for 2 hr at 66,000 X g at 40C, and the virus was resuspended in half the original volume with cold phosphatebuffered saline. Samples of all viruses were kept at -700C. Interferon and Antibody to Interferon. Mouse interferon (MIF) was induced in C243-3 cells with NDV by the method of Tovey et al. (21). Cells were exposed to NDV for 1 hr at 370C at a multiplicity of 10 PFU/ml. Residual virus was removed by rinsing and cells were re-fed with BME/2% FCS. After further incubation at 370C for 24 hr, the culture fluid was harvested, clarified by low-speed centrifugation, and treated with 1 M HCO to bring the pH to 2.0. After further treatment for 5 days at 40C the clarified supernatant containing the MIF was readjusted to pH 7.5 with 1 M NaOH, then centrifuged at 100,000 X g for 1 hr at 40C and filtered through a Millipore 0.22-ym-pore-diameter membrane. Samples of the processed MIF were stored at -70'C. Infectivity tests on monolayers of chicken fibroblasts or in embryonated eggs showed that no infectious NDV was present in the MIF preparations. The preparations satisfied the criteria of Lockart (22) for virusinduced interferons. The antiviral activity of the crude MIFs, when assayed by the procedure of Dahl and Degre (23), closely approximated that of National Institute of Allergy and Infections Diseases reference MIF (no. G002-904-51 1). Partially purified fibroblast MIF and antibody to mouse fibroblast IF (anti-MIF) were gifts from Kurt Paucker, Medical College of Pennsylvania. The MIF had been partially purified by antibody affinity chromatography and titered 6.8 X 105 units/ml. The anti-MIF inhibited 3.8 X 103 reference units of fibroblast MIF per ml. MIF was warmed to 37°C before use. DRB. This chemical was obtained from Calbiochem. It was dissolved in dimethyl sulfoxide, then diluted 1:100 in BME to give a solution containing 1000,g/ml and filtered through an 0.22-,um Millipore membrane. It was diluted in serum or amino-acid-free BME/2% FCS before use at 100 MM final concentration in MIF, anti-MIF, or BME/2% FCS. Incorporation of 13HljUridine into RNA. We used the method devised by Tamm et al. (18) to determine the rapidity and extent of the inhibition of RNA synthesis of L-929 cells exposed to 100 uM DRB and the release of the inhibition after a medium change. After exposure to [3Hjuridine (ICN) at a concentration of 2.5 MCi/ml (1 Ci = 3.7 X 1010 becquerels), cells were washed four times with ice-cold phosphate-buffered saline and the drained monolayers were frozen at -70°C and scraped into 1 ml of water. Each determination was done in tripli-